Diagnostic Microbiology and Infectious Disease

44 (2002) 363–366 www.elsevier.com/locate/diagmicrobio

Comparison of methods of interpretation of checkerboard synergy

testing
Charles R. Bonapace1, John A. Bosso*, Lawrence V. Friedrich, Roger L. White
Anti-Infective Research Laboratory, College of Pharmacy, Medical University of South Carolina, Charleston, South Carolina, USA

Received 5 February 2002; accepted 31 July 2002

Abstract
Four different methods for interpreting the results of checkerboard synergy testing were compared by applying each to a set of synergy
study data. Statistically significant differences in synergy were detected among methods (% synergy ranged from 10 to 83%). As
interpretations were found to vary widely based upon method, one should be aware of this in interpreting the relevant literature. © 2002
Elsevier Science Inc. All rights reserved.

1. Introduction al., 1993; Marymont & Marymont, 1981; Sanders et al.,

It is well recognized that the results of studies evaluating
the nature of antibiotic interactions can vary, depending on
the methodology utilized (Bayer & Morrison, 1984; Cap-
pelletty & Rybak, 1996; Chan & Zabransky, 1987; Klater-
sky et al., 1976; Lerner et al., 1984; Moody et al., 1987;
Norden et al., 1979; Ryan et al., 1981; Weinstein et al.,
1975; White et al., 1996.). It is not surprising that with the
two most common methods, checkerboard and time-kill
testing, results might vary as these techniques measure two
different endpoints: inhibition of growth and killing. Fur-
ther, with the checkerboard technique, conclusions can vary
depending on how one analyzes the experimental results.
While the methodology of conducting a checkerboard ex-
periment is well standardized, a variety of methods for
interpreting the results have been utilized (Anon, 1992;
Berenbaum, 1978; Den Hollander et al., 1998; Eliopoulos,
1989; Hallander et al., 1982; Hamilton-Miller, 1995; Li et
* Corresponding author. Tel.: ⫹843-792-8501; fax: ⫹843-792-1712.
E-mail address: bossoja@musc.edu (J. A. Bosso).
Presented in part at the American Society for Microbiology General
Meeting, Chicago, Illinois May 1999, Abstract #A-87
1
Current Address: U.S. Food and Drug Administration, Center for
Drug Evaluation & Research, Office of Clinical Pharmacology and Bio-
pharmaceutics, Division of Pharmaceutical Evaluation III, 9201 Corporate
Blvd, HFD-880, Rockville, MD 20850.
This paper was written by Charles Bonapace in his private capacity. No
official support or endorsement by the Food and Drug Administration is
intended or should be inferred.
1993; Weinstein et al., 1975). These methods of interpreta-
tion may, in turn, lead to different results and conclusions.
In order to explore this possibility, we utilized four methods
of interpreting checkerboard results to evaluate results from
a previously conducted synergy trial (Bonapace et al.,
2000).
2. Materials and Methods
In our original study, susceptibility testing was per-
formed using the broth microdilution method against 10
clinical non-urine isolates of Acinetobacter baumannii with
Pseudomonas aeruginosa ATCC 27853 as a control strain.
As previously reported, ranges of modal minimum inhibi-
tory concentrations (MIC) for the test isolates to the various
antibiotics as assessed by broth microdilution were as fol-
lows: piperacillin: 8-3072 ␮g/ml, cefepime: 1-96 ␮g/ml,
tobramycin: 0.25-32 ␮g/ml, and trovafloxacin: 0.03-⬎ 4
␮g/ml. Checkerboard synergy testing was performed in
triplicate with 96-well microtiter trays using an 8-by-8 well
configuration. Positive growth controls (to assess the pres-
ence of turbidity) were performed in wells not containing
antibiotic. In addition, negative growth controls were per-
formed in a separate microtiter tray. Combinations tested
consisted of cefepime ⫹ tobramcyin, cefepime ⫹ trova-
floxacin, piperacillin ⫹ tobramycin, and piperacillin ⫹
trovafloxacin. Concentrations tested ranged from 0.031⫻
MIC to 4⫻ MIC of each respective antibiotic. The final
inoculum was approximately 5 ⫻ 105 CFU/mL and verified

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364 C.R. Bonapace et al. / Diagnostic Microbiology and Infectious Disease 44 (2002) 363–366
Figure 1. Schematic of checkerboard methods of interpretation ⵧ Non-turbid well
by method number within each.
in duplicate. Microtiter trays were incubated for 18 h at
35°C and turbidity assessed visually using a lighted micro-
plate reader.
The interpretation of the checkerboard synergy testing
results was determined with the following 4 methods (Fig-
ure 1). Method #1 [Mean Fractional Inhibitory Concen-
tration (FIC) Index]: This method, described by Den Hol-
lander et al. (Den Hollander et al., 1998) was the method
actually used in our previously described synergy study
(Bonapace et al., 2000). The FIC index was calculated using
the concentrations in the first non-turbid (clear) well found
in each row and column along the turbidity/non-turbidity
interface and then averaged. This method does not require
an entire row or column to have complete inhibition of
growth. Method #2 (Full Row/Column): For this method
Turbid well Wells utilized in each method of interpretation are signified
the FIC index was calculated for each drug using the col-
umn and row with the lowest concentrations in which there
was no turbidity (no visual growth) observed in the entire
row and column (Eliopoulos, 1989). Method #3 (Lowest
FIC Index): The lowest FIC index of all the non-turbid
wells along the turbidity/non-turbidity interface was used.
This method is described in the Clinical Microbiology Pro-
cedures Handbook (Anon, 1992). Method #4 (Two Well):
Synergy was defined as the absence of turbidity in the two
wells containing 0.25⫻ MIC of both drugs and 2⫻ MIC of
both drugs, antagonism was defined as the presence of
turbidity in both of these wells whereas, additivity/indiffer-
ence was defined as all other possibilities (Eliopoulos &
Moellering, 1996; Marymont & Marymont, 1981).
For methods 1-3, the FIC was calculated for each anti-
 C.R. Bonapace et al. / Diagnostic Microbiology and Infectious Disease 44 (2002) 363–366 365

Table 1 next highest two-fold dilution if growth occurred in the

Checkerboard results from all four methods outside row or column (highest concentration) of a micro-
Drug A/Drug B Method Method Method Method titer tray. The cumulative FIC index (⌺FIC) was calculated
#1 #2 #3 #4 for each antimicrobial combination as the sum of the indi-
Trovafloxacin/Cefepimea vidual FICs. The ⌺FIC from each microtiter tray was cal-
Median FIC Index 0.4 1.0 0.3 NAb culated and the average of the three replicates used in
Range (0.2–1.1) (0.5–8.5) (0.2–0.3) NAb subsequent analyses. Synergy was defined as an ⌺FIC ⱕ0.5,
% Synergy 60 20 100 100 additivity/indifference was defined as an ⌺FIC ⬎0.5 to 4,
% Additivity/Indifference 40 60 0 0
and antagonism was defined as an ⌺FIC ⬎4 (Eliopoulos &
% Antagonism 0 20 0 0
Moellering, 1996). ␹2 analysis was used to assess differ-
Trovafloxacin/Piperacillina ences among the 4 methods of categorical interpretation
Median FIC Index 0.6 1.4 0.3 NAb utilizing all drug combinations. Additionally, ␹2 analysis
Range (0.2–0.7) (0.5–1.7) (0.2–0.4) NAb
was used to assess differences among categorical interpre-
% Synergy 40 20 100 80
% Additivity/Indifference 60 80 0 20 tations using the 4 methods for each individual drug com-
% Antagonism 0 0 0 0 bination. Statistical significance was defined a priori as p
ⱕ0.05. Furthermore, we calculated percentage agreement
Tobramycin/Cefepime
between each of these four methods of checkerboard inter-
Median FIC Index 0.8 1.8 0.5 NAb
Range (0.2–1.4) (0.6–3.5) (0.1–0.6) NAb pretation with the results from the time-kill studies from our
% Synergy 30 0 70 40 original study (Bonapace et al., 2000).
% Additivity/Indifference 70 100 30 60
% Antagonism 0 0 0 0
3. Results
Tobramycin/Piperacillin
Median FIC Index 0.6 1.5 0.3 NAb
Range (0.2–1.2) (0.4–4.0) (0.1–1.0) NAb
The results of synergy testing by drug combination and
% Synergy 40 10 80 80 method of interpretation are displayed in Table 1. Interpre-
% Additivity/Indifference 60 90 20 20 tation varied widely among the methods. Among these four
% Antagonism 0 0 0 0 methods of interpretation, the rank order of increasing like-
a
n ⫽ 5 isolates lihood of detecting synergy was method #2 (10%), method
b
NA - not applicable to this method #1 (40%), method #4 (70%), and method #3 (83%, p
⬍0.0001). A result or interpretation of antagonism was
determined once, occurring with method #2 (full row/col-
microbial used in combination as the MIC of the drug in umn). Differences among the method of interpretation were
combination divided by the MIC of that drug when tested also noted for each drug combination (p ⱕ0.0396) with the
alone. The MIC of a drug in combination was rounded to the exception of trovafloxacin/cefepime (p ⫽ 0.0730). Of inter-

Figure 2. Examples of differences among the methods of interpretation.

366 C.R. Bonapace et al. / Diagnostic Microbiology and Infectious Disease 44 (2002) 363–366
est, the interpretive method most likely to concur with the
time-kill results from our original study was the second (full
row/column method) that agreed 43-80% of the time de-
pending on the concentration studied in the time-kill test.
The poorest agreement was with method #1 and time-kill
tests using 2⫻ MIC of both antibiotics (15%). Whether this
high degree of agreement between time-kill and method #2
was influenced by the fact that little synergy or antagonism
was detected is unknown.
4. Discussion
Occurrence of synergy using the checkerboard technique
appears to be highly dependent on the method of interpre-
tation. In what manner interpretations would vary with the
four methods used here are illustrated with three sets of
sample data in Figure 2. One should be aware of this
possibility when reading and interpreting the associated
literature. It must also be emphasized that many other meth-
ods of checkerboard result interpretation beyond those ex-
amined here can be found in the literature. Moreover, the
reader is often unable to ascertain the method of interpre-
tation in studies using the checkerboard technique. This
variability in technique may partially explain discordant
results from studies reporting antibiotic interactions, appar-
ently using identical methodology. Standardization of inter-
pretation would be desirable. Finally, correlation of these
methods with outcomes from clinical studies is needed to
determine the most useful method.
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